DNA and protein synthesis in cultured lens epithelial cells. I. Test system]

An in vitro test system for measuring DNA and protein synthesis in cultivated lens epithelium cells was developed. The method is suited also for other monolayer cultures; it has the following advantages: a) Cultivation of cells, incubation with radionuclides, preparation of the samples and measurement of radioactivity are carried out in the same vessel (scintillation vial); b) The use of 3H-thymidine and 14C-phenylalanine allows simultaneous measurement of DNA and protein synthesis; c) Only small amounts of cells (10(4) to 10(5) cells) are required to measure DNA and protein synthesis. The test system is highly sensitive to synthetic effectors (cytosone arabinoside, actinomycin D, puromycin), and is thus appropriate for the detection of inhibitors of DNA and protein synthesis and for testing the toxicity of drugs.     

A novel in vitro system, the integrated discrete multiple organ cell culture (IdMOC) system, for the evaluation of human drug toxicity: comparative cytotoxicity of tamoxifen towards normal human cells from five major organs and MCF-7 adenocarcinoma breast cancer cells

In vitro assays involving primary cells are used routinely to evaluate organ-specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity. A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism. A novel cell culture system, the integrated discrete multiorgan cell culture system (IdMOC), is described here. The IdMOC is based on the "wells within a well" concept, consisting of a cell culture plate with larger, containing wells, within each of which are multiple smaller wells. Cells from multiple organs can be cultured initially in the small wells (one organ per well, each in its specialized medium). On the day of toxicity testing, a volume of drug-containing medium is added to the containing well to flood all inner wells, thereby interconnecting all the small wells. After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate endpoints. We report here the application of IdMOC in the evaluation of the cytotoxicity of tamoxifen, an anticancer agent with known human toxicity, on primary cells from multiple human organs: liver (hepatocytes), kidney (kidney cortical cells), lung (small airway epithelial cells), central nervous system (astrocytes), blood vessels (aortic endothelial cells) as well as the MCF-7 human breast adenocarcinoma cells. IdMOC produced results that can be used for the quantitative evaluation of its anticancer effects (i.e., cytotoxicity towards MCF-7 cells) versus its toxicity toward normal organs (i.e., liver, kidney, lung, CNS, blood vessels).     

Integrated decision-tree testing strategies for developmental and reproductive toxicity with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME conducted a research project, sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for the use of alternative methods (both in vitro and in silico) in developmental and reproductive toxicity testing. It considers many tests based on primary cells and cell lines, and the available expert systems and QSARs for developmental and reproductive toxicity, and also covers tests for endocrine disruption. Ways in which reduction and refinement measures can be used are also discussed, particularly the use of an enhanced one-generation reproductive study, which could potentially replace the two-generation study, and therefore considerably reduce the number of animals required in reproductive toxicity. Decision-tree style integrated testing strategies are also proposed for developmental and reproductive toxicity and for endocrine disruption, followed by a number of recommendations for the future facilitation of developmental and reproductive toxicity testing, with respect to human risk assessment.     

Minireview: Apoptosis as seen through a lens

The lens represents an ideal model system for studying many of the cellular and molecular events of differentiation. It is composed of two ectodermally-derived cell types: the lens epithelial cells and the lens fibre cells, which are derived from the lens epithelial cells by differentiation. Programmed removal of nuclei and other organelles from the lens fibre cells ensures that an optically clear structure is created, while the morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. These observations suggest the existence of biochemical parallels between the process of lens fibre cell organelle loss and classical apoptosis. For example, proteins encoded by the bcl-2 and caspase gene families are expressed in developing lenses and nuclear degeneration in lens fibre cells can be inhibited in vivo by overexpression of bcl-2 and in vitro by incubation of differentiating lens epithelial cell cultures with caspase inhibitors. Thus, the developing lens may represent a particularly useful model system for researchers interested in apoptosis. In this review, the recent literature pertaining to lens fibre cell organelle loss and its relationship to apoptosis is reviewed and possible future research directions are suggested.     

Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration

The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens-specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.     

Molecular targets and early response biomarkers for the prediction of developmental toxicity in vitro

There is an urgent need for new in vitro methods to predict the potential developmental toxicity of candidate drugs in the early lead identification and optimisation process. This would lead to a reduction in the total number of animals required in full-scale developmental toxicology studies, and would improve the efficiency of drug development. However, suitable in vitro systems permitting robust high-throughput screening for this purpose, for the most part, remain to be designed. An understanding of the mechanisms involved in developmental toxicity may be essential for the validation of in vitro tests. Early response biomarkers - even a single one - could contribute to reducing assay time and facilitating automation. The use of toxicogenomics approaches to study in vitro and in vivo models in parallel may be a powerful tool in defining such mechanisms of action and the molecular targets of toxicity, and also for use in finding possible biomarkers of early response. Using valproic acid as a model substance, the use of DNA microarrays to identify teratogen-responsive genes in cell models is discussed. It is concluded that gene expression in P19 mouse embryocarcinoma cells represents a potentially suitable assay system, which could be readily used in a tiered testing system for developmental toxicity testing.     

Application of the new ccdAB-type natural toxin-antitoxin module for stabilization of inheritance of expressive plasmid vectors based on the bacteriophage N15 replicon in Escherichia coli cells

Biopharmaceutical industry currently produces considerable quantity of novel recombinant preparations by way of overexpression in Escherichia coli cells, an inexpensive, efficient, time-proven, and practically feasible system of heterologous expression. Due to the instability of maintenance and inheritance of expression vectors in producer cells, the cells that have spontaneously lost the plasmid gain a significant selective advantage over the cells producing a heterologous protein and accumulate in the fermentor. For solution of this problem, it is proposed to develop a new generation of expression vectors with high stability of inheritance in the absence of external selective pressure, using a replicon of phage N15, which possesses its own system for active distribution of plasmid copies in the daughter cells, supplemented by a toxin-antitoxin genetic module preventing the loss of a plasmid. Two new addiction modules homologous to the known ccdAB and mazEF systems were isolated from natural enterobacterial populations and characterized. The testing showed more effective operation of the ccdAB module. The latter was a basis for construction of new expression vectors pN15E41 and pN15E61 demonstrating the high synergism of action of the plasmid segregation systems and the addiction module and directly applicable for biotechnological practice.     

Lens formation by pigmented epithelial cell reaggregate from dorsal iris implanted into limb blastema in the adult newt

In newt lens regeneration, the dorsal iris has lens forming ability and the ventral iris has no such capability, whereas there is no difference in the morphological criteria. To investigate the real aspects of this characteristic lens regeneration in the newt at the cellular level, a useful model system was constructed by transplanting the dorsal and ventral reaggregate derived from singly dissociated pigmented epithelial cells of the iris into the blastema of the forelimb in the newt. The lens was formed from the dorsal reaggregate with high efficiency, but not from the ventral one. No lens formation was observed in the implantation of the reaggregate into the tissue of the intact limbs. In detailed examination of the process of lens formation from the reaggregate, it was shown that tubular formation was the first step in the rearrangement of cells within the reaggregate. This was followed by depigmentation, vesicle formation with active cell growth, and the final step was lens fiber formation by transdifferentiation of epithelial cells composing the lens vesicle. The process was almost the same as in situ lens regeneration except the reconstitution of the two-layered epithelial structure was embodied as flattened tubular formation in the first step. The present study made it possible for the first time to examine lens forming ability in the reaggregate mixed with dorsal and ventral cells, because the formation of a reaggregate was started from singly dissociated cells of the dorsal and ventral cells of the iris. Mixed reaggregate experiments indicated that the existence of the dorsal cells in a cluster within the reaggregate is important in lens formation, and ventral cells showed an inhibitory effect on the formation. The present study demonstrated that the limb system thus constructed was effective for the analysis of lens formation at the cellular level and made it possible to examine the role of dorsal and ventral cells in lens regeneration.     

Environmental impact of thin-film GaInP/GaAs and multicrystalline silicon solar modules produced with solar electricity

Background, aim, and scope  The environmental burden of photovoltaic (PV) solar modules is currently largely determined by the cumulative input of fossil energy used for module production. However, with an increased focus on limiting the emission of CO₂ coming from fossil fuels, it is expected that renewable resources, including photovoltaics, may well become more important in producing electricity. A comparison of the environmental impacts of PV modules in case their life cycle is based on the use of PV electricity in contrast to conventional electricity can elucidate potential environmental drawbacks in an early stage of development of a solar-based economy. The goal of this paper is to show for ten impact categories the environmental consequences of replacing fossil electricity with solar electricity into the life cycle of two types of PV modules. Materials and methods  Using life cycle assessment (LCA), we evaluated the environmental impacts of two types of PV modules: a thin-film GaInP/GaAs tandem module and a multicrystalline silicon (multi-Si) module. For each of the modules, the total amount of fossil electricity required in the life cycle of the module was substituted with electricity that is generated by a corresponding PV module. The environmental impacts of the modules on the midpoint level were compared with those of the same modules in case their life cycle is based on the use of conventional electricity. The environmental impacts were assessed for Western European circumstances with an annual solar irradiation of 1000 kWh/m². For the GaInP/GaAs module, the environmental impacts of individual production steps were also analysed. Results  Environmental burdens decreased when PV electricity was applied in the life cycle of the two PV modules. The impact score reductions of the GaInP/GaAs module were up to a factor of 4.9 (global warming). The impact score reductions found for the multi-Si module were up to a factor of 2.5 (abiotic depletion and global warming). Reductions of the toxicity scores of both module types were smaller or negligible. This is caused by a decreased use of fossil fuels, on the one hand, and an increased consumption of materials for the production of the additional solar modules used for generating the required PV electricity on the other. Overall, the impact scores of the GaInP/GaAs module were reduced more than the corresponding scores of the multi-Si module. The contribution analysis of the GaInP/GaAs module production steps indicated that for global warming, the cell growth process is dominant for supply with conventional electricity, while for the solar scenario, the frame becomes dominant. Regarding freshwater aquatic ecotoxicity scores associated with the life cycle of the GaInP/GaAs module, the cell growth process is dominant for supply with conventional electricity, while the reactor system for the cell growth with the associated gas scrubbing system is dominant for the solar scenario. Discussion  There are uncertainties regarding the calculated environmental impact scores. This paper describes uncertainties associated with the used economic allocation method, and uncertainties because of missing life cycle inventory data. For the GaInP/GaAs module, it was found that the global warming impact scores range from −66% to +41%, and the freshwater aquatic ecotoxicity scores (for an infinite time horizon) range from −40% to +300% compared to the default estimates. For both impact categories, the choices associated with the allocation of gallium, with the electricity mix, with the conversion efficiency of the commercially produced GaInP/GaAs cells, and with the yield of the cell growth process are most influential. For freshwater aquatic ecotoxicity, the uncertainty concerning the lifetime of the reactor system for the GaInP/GaAs cell growth process and the gas scrubbing system is particularly relevant. Conclusions  Use of PV electricity instead of fossil electricity significantly reduces the environmental burdens of the GaInP/GaAs and the multi-Si module. The reductions of the toxicity scores, however, are smaller or negligible. Toxicity impacts of the GaInP/GaAs cells can be reduced by improvement of the yield of the cell growth process, a reduced energy demand in the cell growth process, reduction of the amount of stainless steel in the cell growth reactor system and the gas scrubbing system, and a longer lifetime of these systems. Recommendations and perspectives  Because the greenhouse gas emissions associated with the production of fossil-fuel-based electricity have an important share in global warming on a world-wide scale, switching to a more extensive use of solar power is helpful to comply with the present international legislation on the area of global warming reduction. As reductions in toxicity impact scores are smaller or negligible when fossil electricity is replaced by PV electricity, it is desirable to give specific attention to the processes which dominantly contribute to these impact categories. Furthermore, in this study, a shift in ranking of several environmental impacts of the modules has been found when PV electricity is used instead of fossil electricity. The results of a comparative LCA can thus be dependent of the electricity mix used in the life cycles of the assessed products. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.